Method of stabilizing heme protein and storage solution therefor

ABSTRACT

A method of stabilizing a hem protein which is effective against the denaturation and degradation of a hem protein typified by hemoglobin and a storage solution therefor. A method of stabilizing a hem protein and a storage solution therefor characterized in that an iminocarboxylic acid or its salt is made to coexist in a sample containing the hem protein, wherein the above-described iminocarboxylic acid is a compound represented by the following general formula (1) wherein R represents a hydrogen atom or a hydroxyl group; and X&#39;s represent each a hydrogen atom, an alkali metal or an ammonium group.

TECHNICAL FIELD

The present invention relates to a method of stabilizing a heme protein,particularly to a method of stabilizing a heme protein in a heme proteintest of feces, urine, or a blood sample.

BACKGROUND ART

In recent years, detections of fecal human hemoglobin (fecal occultblood) caused by bleeding from gastrointestinal tracts have been broadlycarried out as a screening method of a digestive system disorder, suchas colon cancer. The detective method of human hemoglobin is animmunological method employing a specific antibody against humanhemoglobin, which takes the place of a related-art dipstick method basedon a chemical coloring reaction. This method does not require a dietaryrestriction, and therefore it becomes established as a convenienttesting method.

Examples of an immunological detective method for human hemoglobininclude: a single immunodiffusion test in agar plates employing aprecipitation line between an anti-human hemoglobin antibody and a humanhemoglobin in a testing sample; a latex agglutination test using a latexparticle sensitized with an anti-human hemoglobin antibody; enzymeimmunoassay or radioimmunoassay, using an anti-human hemoglobin antibodylabeled with an enzyme or a radioactive element; and gold colloidaggregation calorimetric method using a gold colloid particle sensitizedwith an anti-human hemoglobin antibody.

However, in a testing solution, human hemoglobin is gradually denatured,and its antigenicity is decreased. Also, in a testing solution, storageconditions such as storage temperature often accelerates denaturation ofhuman hemoglobin, or bacteria and digestive enzymes in feces oftendegrade human hemoglobin. Such denaturation and degradation destroyhuman hemoglobin conformation, resulting in decrease of antigenicity.Therefore, in an immunological method for measuring human hemoglobin,denaturation and degradation of the hemoglobin involve an incorrectdiagnosis.

Meanwhile, on a fecal occult blood test, feces are often collected bysubjects themselves at their places, and are provided for the test witha closed container, by dissolving the feces in a feces dissolvingsolution in the container. In such cases, human hemoglobin in feces isoften remained in the solution for several days, or placed in a hightemperature when utilizing a transportation method such as a postalservice. Also, even when feces are collected in a clinical laboratory,it sometime takes longer until carrying out the fecal occult blood testbecause other tests are also performed. Under such circumstances, anaccurate measurement is disturbed by denaturation and degradation ofhuman hemoglobin as described above.

To prevent such denaturation and degradation of human hemoglobin in asolution, a method of adding common antibacterial agents such asthimerosal and chlorhexidine (see Patent Document 1, for example); amethod of adding saccharides (see Patent Document 2, for example);addition of hemoglobin of animals other than human (see Patent Document3, for example); addition of sera of animals other than human (seePatent Document 4, for example); addition of a bacteriolytic enzyme (seePatent Document 5, for example); addition of iron protoporphyrin (seePatent Document 6, for example) and the like have been developed.

However, the techniques for stabilizing human hemoglobin described inthose publications cannot fully suppress denaturation and degradation ofhuman hemoglobin in a testing solution containing feces.

Other than those described above, a method of stabilizing hemoglobinusing ethylenediaminetetraacetic acid (hereafter, abbreviated as EDTA)have been developed (see Patent Document 7, for example).

However, as a result of replication studies conducted by the inventorsof the present invention, it has been confirmed that a sufficientstabilizing action on fecal human hemoglobin cannot be expected with asingle use of EDTA.

Then, the applicant of the present invention has been developing amethod of adding an aqueous transition metal complex which is moreeffective for stabilization than EDTA only (see Patent Document 8, forexample). Further, the applicant has already developed a method ofstabilizing hemoglobin with which a ferrocyanide compound coexists (seePatent Document 9, for example); a method of stabilizing hemoglobin withwhich an enzymatic degradation product of hemoglobin coexists (seePatent Document 10, for example); a method of stabilizing a heme proteinwith which transition metals coexist (see Patent Document 11, forexample); a method of stabilizing a heme protein with which an organicacid such as malic acid coexists (see Patent Document 12, for example);and a method of stabilizing a heme protein with which a delipidatedalbumin coexists (see Patent Document 13, for example).

Patent Document 1: Japanese Patent Application Publication No.JP-A-63-271160Patent Document 2: Japanese Patent Application Publication No.JP-A-63-243756Patent Document 3: Japanese Patent Application. Publication No.JP-A-2-296149Patent Document 4: Japanese Patent Application Publication No.JP-A-4-145366

Patent Document 5: Japanese Examined Patent Application Publication No,JP-A-5-69466

Patent Document 6: Japanese Patent Application Publication No.JP-A-5-281227Patent Document 7: Japanese Patent Application Publication No.JP-A-5-99923Patent Document 8: Japanese Patent Application Publication No.JP-A-7-229902Patent Document 9: Japanese Patent Application Publication No.JP-A-11418806Patent Document 10: Japanese Patent Application Publication No.JP-A-11-218533Patent Document 11: Japanese Patent Application Publication No.JP-A-2001-249132Patent Document 12: Japanese Patent Application Publication No.JP-A-2003-014768Patent Document 13: Japanese Patent Application Publication No.JP-A-2003-194825

DISCLOSURE OF THE INVENTION Problem to be Solved by the Invention

It is a first object of the present invention to provide a novel methodof stabilizing a heme protein, which is effective against denaturationand degradation of a heme protein such as hemoglobin. In particular, thepurpose of the invention is to provide a technique for effectivelystabilizing hemoglobin which coexists with feces components havingstrong denaturation and degradation activities.

It is a second object of the present invention to provide a storagesolution of a heme protein, which employs a novel stabilizing agent fora heme protein.

It is a third object of the present invention to provide a specificcompound for use in a novel method of stabilizing a heme protein.

Means for Solving the Problem

The present invention includes following (1) to (7).

(1) A method of stabilizing a heme protein is characterized by includingallowing an iminocarboxylic acid or a salt thereof to coexist in asample containing the hems protein.(2) In the method of stabilizing a heme protein according to (1), theiminocarboxylic acid or a salt thereof is a compound of Formula (1);

(where R is a hydrogen atom or a hydroxy group, and X is a hydrogenatom, an alkali metal, or an ammonium group), or a mixture thereof.(3) In the method of stabilizing a hems protein according to (1) or (2),the iminocarboxylic acid or a salt thereof is3-hydroxy-2,2′-iminodisuccinic acid of Formula (2):

(where X is a hydrogen atom, an alkali metal, or an ammonium group), ora salt thereof.(4) In the method of stabilizing a heme protein according to any one of(1) to (3), a concentration of the iminocarboxylic acid or a saltthereof falls within a range of 0.001 to 0.200 mol/L.(5) In the method of stabilizing a home protein according to (1), theheme protein is hemoglobin, myoglobin, peroxidase, or catalase.(6) In the method of stabilizing a heme protein according to (1), asample containing the heme protein is feces, urine, or blood.(7) A storage solution for a heme protein contains the iminocarboxylicacid or a salt thereof according to any one of (1) to (3).(8) An iminocarboxylic acid or a salt thereof used in the method ofstabilizing a heme protein according to any one of (1) to (6).

EFFECTS OF THE INVENTION

In the method of stabilizing a heme protein of the present invention,the heme protein can be protected from a biogenic substance havingstrong denaturation and degradation activities, even under coexistencewith a feces component. Accordingly, the method is useful to stabilizehemoglobin which is contained in a sample for analysis of a biogenicsubstance, for example, for detection of fecal occult blood. Especially,the method contributes to maintain antigenicity of a heme protein, whenthe heme protein is supposed to be immunologically analyzed and itsantigen structure is required to be protected. By the compound used forthe method of the present invention, the storage solution, and themethod of stabilizing a heme protein, hemoglobin in samples of feces isstabilized effectively, and prevention of a false negative resultcaused, by denaturation and degradation of hemoglobin can be expected.

BEST MODES FOR CARRYING OUT THE INVENTION

The present invention will be described below in more detail.

In the present invention, the iminocarboxylic acid or a salt thereof iscoexisted with a heme protein in a sample. The iminocarboxylic acid canbe appropriately selected from ones well known in the art; however,especially the compound of Formula (1) is preferable, and3-hydroxy-2,2′-iminodisuccinic acid of Formula (2) or a salt thereof ismore preferable. Also, examples of an alkali metal of X of Formula (1)and Formula (2), include lithium, sodium, potassium, rubidium, andcesium.

Also, as the iminocarboxylic acid used in the present invention, forexample, ones synthesized by a method using aspartic acid andepoxysuccinic acid as raw materials (Japanese Patent ApplicationPublication No. JP-A-9-183773, for example); a method using maleic acidand ammonia as raw materials (U.S. Pat. No. 639,863, for example); amethod using aspartic acid and maleic acid as raw materials (JapanesePatent Application Publication No. JP-A-5-320109, for example); a methodusing maleic acid half ester; maleic acid and ammonia as raw materials(Japanese Patent Application Publication No. JP-A-8-12631, for example)can be used.

A concentration of the iminocarboxylic acid or a salt thereof in asample containing a hems protein is 0.001 to 0.200 mol/L, morepreferably, 0.050 to 0.150 mmol/L. When the concentration of theiminocarboxylic acid or a salt thereof is less than 0.001 mmol/L, aneffect of stabilizing a heme protein becomes insufficient. On the otherhand, when the concentration of the iminocarboxylic acid or a saltthereof is over 0.200 mmol/L, solubility decreases, and animmunoreaction is also inhibited.

Although it is not essential in the present invention, by adding a knownprotein protectant as required, an effect to stabilize the heme proteincan be further enhanced. Examples of such a protein protectant includean inactive protein such as albumin and gelatin.

Any albumin can be used; however, albumin derived from animal serum oran egg is especially preferable. Specific examples thereof includealbumin derived from blood of cattle, horses, goats, sheep, pigs,rabbits, and cubs or fetuses thereof.

In addition to the albumin described in above, an enzymatic degradationproduct thereof is also known. As albumin of the present invention, aprotein induced from such albumin can also be included.

In addition to the above protein protectant, addition of various knownprotein protective components for a heme protein, such as anantibacterial agent to prevent unnecessary growth of a microorganism,and a buffer giving pH favorable for preserving the heme protein is alsoeffective.

Examples of an antibacterial agent include a series of non-penicillinantibiotics, in addition to bacteriolytic enzyme, ethyl benzoate,penicillin, Fungizone, streptomycin, and cephamycin. Also, it is knownthat protease inhibitors such as trypsin inhibitor and alpha-2macroglobulin also stabilizes the heme protein.

A pH of a dispersion medium is adjusted to a range which allows a hemeprotein to be stably maintained. Under extremely acidic or alkalineconditions, stabilization of the heme protein may be lost, and thereforeneutral pH is preferable. Concretely, the pH is 5 to 10, preferablyabout 6 to 8.

An appropriate buffer can be employed to maintain the pH. For example,the Good's buffer such as hydroxyethylpiperazine-2-ethanesulfonic acid(hereafter, abbreviated as HEPES) and piperazine-bis(2-ethanesulfonicacid) (hereafter, abbreviated as PIPES), gives pH 6 to 8 which may bethe most appropriate pH to stabilize a structure of the heme protein.Moreover, the Good's buffer is also utilized as a reaction buffersolution to detect the heme protein by an immune reaction, and it can belisted as an especially preferable buffer. In addition, a phosphatebuffer solution, a Tris buffer solution, a glycine buffer solution, aboric acid buffer solution, and the like can also be employed.

The method of stabilizing a heme protein of the present invention can beemployed to detect the heme protein in a feces, urine, or blood sample.The heme protein can be appropriately selected from proteins having hemas their components. Examples thereof include hemoglobin, myoglobin,peroxidase, or catalase. Especially, it contributes to maintainantigenicity of a heme protein, when the heme protein is immunologicallyanalyzed and its antigen structure is required to be protected.

The present invention can provide a storage solution for hemoglobincontaining an iminocarboxylic acid or salts thereof which act(s) as astabilization factor of a lime protein.

In addition, the storage solution of the present invention can contain aprotein protectant or a buffer as needed. The protein protectant or thebuffer can be appropriately selected from the ones listed in the above.

The present invention can provide an immunological measurement methodfor a heme protein as an application of the stabilizing technique of aheme protein. Examples of the immunological measurement method include alatex agglutination reaction method, a gold colloid aggregation reactionmethod, immunochromatography, and ELISA. In any measurement methods,antigen activity during storage is maintained by coexistence of animinocarboxylic acid or salts thereof and a heme protein in a sample,and decrease of measurement value is prevented.

The method of stabilizing a heme protein of the present invention isuseful to stabilize heme protein as an analytical object which exists ina biogenic sample. Especially in a heme protein measurement methodutilizing an immunological procedure with a heme protein recognitionantibody, antigenicity of a heme protein as an analytical object ishighly stabilized. Feces, urine, and blood are known as biogenic samplesto detect a heme protein. Especially, hemoglobin in feces will be anindicator of bleeding in a gastrointestinal system, and hemoglobin inurine will be an indicator of bleeding in a urinary tract.

When applying the stabilization method of the present invention tohemoglobin in a sample of feces, it is recommended to add animinocarboxylic acid or salts thereof in a storage solution in whichfeces are suspended. Usually, in the detection of hemoglobin in feces,the feces are suspended in an appropriate storage solution, and a samplefor an immunological analysis will be prepared by filtration as needed.

Hereinafter, the present invention will be described more in detailreferring to Examples which should not be construed as limiting thescope of the present invention.

EXAMPLES Example Hemoglobin Stabilization Effect of HIDS of DifferentConcentrations

A storage solution was prepared by adding 0 to 0.2 mol/L of tetrasodium3-hydroxy-2,2′-iminodisuccinate (HIDS) of various concentrations shownin Table 1 to a solution (pH 7.0) containing 0.05 mol/L HEPES, 0.9%sodium chloride, and pure water as remainder. The HIDS manufactured byNippon Shokubai Co., LTD. was used.

Hemolyzed hemoglobin, or a mixture of hemolyzed hemoglobin and a fecessample was added to the storage solution, and each test solution wasmeasured for a hemoglobin concentration (concentration immediately afteraddition). Subsequently, the hemoglobin concentration of the testsolution stored at 37° C. for 20 hours was divided by the concentrationimmediately after addition, and residual ratio of hemoglobin wascalculated.

A concentration of hemoglobin was measured by OC-SENSOR NEO analyzer(manufactured by Men Chemical Co., Ltd.) using OC Auto III (manufacturedby Eiken Chemical Co., Ltd.), which is an immunological measurementreagent for hemoglobin.

As shown in Table 1, in the test solution in which HIDS is added to onlyhemoglobin, a stabilizing effect was observed by addition of HIDS whoseconcentration is 0.001 mol/L or more. Also, in the test solution inwhich hemoglobin and feces are coexisted, the stabilizing effect isobserved by addition of HIDS whose concentration is 0.005 mol/L or more.Especially, when a concentration of HIDS added is 0.050 mol/L or more,the effect to stabilize hemoglobin was remarkable.

TABLE 1 Hemoglobin stabilization, effect of HIDS Antigen Hemoglobin +Concentration of HIDS Hemoglobin feces sample added [mol/L] Residualratio [%] Residual ratio [%] — 77.7 27.5 0.001 82.8 28.8 0.005 83.9 32.30.010 84.2 39.2 0.050 84.1 50.8 0.075 85.2 53.9 0.100 87.8 55.9 0.15088.2 57.8 0.200 86.2 46.3

Comparative Example

An effect to stabilize hemoglobin was examined by a method similar tothe method of Example, except for adding ethylenediamine-N,N,N′,N′-4acetic acid (EDTA), iminodiacetic acid (IDA) or N-(2-hydroxyethyl)iminodiacetic acid (HIDA), instead of tetrasodiun3-hydroxy-2,2′-iminodisuccinate (HIDS) of Example.

As shown in Table 2, when EDTA, IDA, or HIDA was used as a chelatingagent, a slight stabilizing effect was observed in the test solution ofhemoglobin only. However, when IDA or HIDA was used as a chelatingagent, any certain stabilizing effect was not observed in the testsolution in which hemoglobin and feces are coexisted. When EDTA wasused, a slightly higher effect to stabilize hemoglobin was observed evenunder coexistence of feces, however, when an concentration of HIDS addedwas 0.010 mol/L or more, the effect to stabilize hemoglobin was muchsmaller than the effect of the storage solution of Example.

TABLE 2 Hemoglobin stabilization effect of various chelating agentsHemoglobin + Hemoglobin feces sample Antigen Residual ratio [%] Residualratio [%] No chelating agent added 77.7 27.5 Concentration of EDTA added[mol/L] 0.001 79.5 34.3 0.010 79.8 35.8 0.050 82.4 37.1 Concentration ofIDA added [mol/L] 0.001 82.1 28.0 0.010 80.9 27.3 0.050 81.0 26.3Concentration of HIDA added [mol/L] 0.001 80.3 26.1 0.010 79.3 26.40.050 81.6 27.3

INDUSTRIAL APPLICABILITY

According to the present invention, when an iminocarboxylic acid or asalt thereof is coexisted with a heme protein in a sample, the hemeprotein can be maintained stably, and therefor; a false negative causedby denaturation and degradation of hemoglobin can be prevented on afecal occult blood test. As a result, a fecal occult blood test can beperformed more accurately.

1. A method of stabilizing a heme protein, the method comprising:allowing an iminocarboxylic acid or a salt thereof to coexist in asample containing the heme protein.
 2. The method of stabilizing a hemeprotein according to claim 1, wherein the iminocarboxylic acid or a saltthereof is a compound of Formula (1):

(where R is a hydrogen atom or a hydroxy group, and X is a hydrogenatom, an alkali metal, or an ammonium group), or a mixture thereof. 3.The method of stabilizing a heme protein according to claim 1, whereinthe iminocarboxylic acid or a salt thereof is3-hydroxy-2,2′-iminodisuccinic acid of Formula (2):

(where X is a hydrogen atom, an alkali metal, or an ammonium group), ora salt thereof.
 4. The method of stabilizing a heme protein according toclaim 1, wherein a concentration of the iminocarboxylic acid or a saltthereof falls within a range of 0.001 to 0.200 mol/L.
 5. The method ofstabilizing a heme protein according to claim 1, wherein the hemeprotein is hemoglobin, myoglobin, peroxidase, or catalase.
 6. The methodof stabilizing a heme protein according to claim 1, wherein a samplecontaining the heme protein is feces, urine, or blood.
 7. A storagesolution for a heme protein comprising the iminocarboxylic acid or asalt thereof according to claim
 1. 8. An iminocarboxylic acid or a saltthereof used in the method of stabilizing a heme protein according toclaim 1.